Role of arginine residue in saccharopine dehydrogenase (L-lysine forming) from baker's yeast.
The baker's yeast saccharopine dehydrogenase (EC 1.5.1.7) was inactivated by 2,3-butanedione following pseudo-first-order reaction kinetics. The pseudo-first-order rate constant for inactivation was linearly related to the butanedione concentration, and a value of 7.5 M-1 min-1 was obtained for the second-order rate constant at pH 8.0 and 25 degrees C. Amino acid analysis of the inactivated enzyme revealed that arginine was the only amino acid residue affected. Although as many as eight arginine residues were lost on prolonged incubation with butanedione, only one residue appears to be essential for activity. The modification resulted in the change in Vmax, but not in Km, values for substrates. The inactivation by butanedione was substantially protected by L-leucine, a competitive analogue of substrate lysine, in the presence of reduced nicotinamide adenine dinucleotide (NADH) and alpha-ketoglutarate. Since leucine binds only to the enzyme-NADH-alpha-ketoglutarate complex, the result suggests that an arginine residue located near the binding site for the amino acid substrate is modified. Titration with leucine showed that the reaction of butanedione also took place with the enzyme-NADH-alpha-ketoglutarate-leucine complex more slowly than with the free enzyme. The binding study indicated that the inactivated enzyme still retained the capacity to bind leucine, although the affinity appeared to be somewhat decreased. From these results it is concluded that an arginine residue essential for activity is involved in the catalytic reaction rather than in the binding of the coenzyme and substrates.
D000596:Amino Acids / Q000032:analysis; D001120:Arginine; D003931:Diacetyl / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012443:Saccharopine Dehydrogenases / Q000378:metabolism*
D000596:Amino Acids; D001120:Arginine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012443:Saccharopine Dehydrogenases; D003931:Diacetyl; D008239:Lysine